作者希望认识到，发展援助和进行免疫组化米歇尔巴内斯的技术援助以及佩特里 - 汉森博士，博士鳗鱼和Timothy Brown评论。该项目是由美国农业部（USDA）CRIS项目＃MISV - 0801310和密西西比州立大学兽医学院。

<html> 1。细菌的挑战<ol><li>细菌在一夜之间成长为30 ° C摇脑心浸液肉汤。 </li><li>停止在鱼缸水的循环和添加肉汤100 1浓度（每升10毫升，例如）。 </li><li>一小时后孵化，在坦克重新启动的循环水，其余的细菌冲洗出来。 </li></ol> 2。采样<ol><li>采样时间和器官取决于研究的目的，但在我们的研究中，我们使用以下采样点:1，6，16，24，36，48，60，72，96，110，125和175小时后感染。 </li><li>在每一个时间点，安乐死六个鱼浸泡在水含MS222和样品（采样机关的选择是对什么是已知的感染过程中的）以下机关:鳃，沿侧线，肠，脾后的肌肉，肝，胃，心，头肾和躯干肾。 </li><li>采样后，立即组织学磁带加载机关和两个小时在1％福尔马林浸泡。 </li><li>固定两个小时后，从福尔马林50％的乙醇，在那里，直到嵌入保持磁带转移。 </li><li>石蜡嵌入机关通宵使用纸巾- Tek的贵宾3000组织的处理器（樱花TEK，托兰斯，加利福尼亚州）和部分在5μm的，使用了Leica RM2255 microtone（徕卡显微系统，班诺克本，伊利诺斯州）。 </li></ol> 3。切片脱蜡<ol><li>沉浸在明确的仪式，让所有的蜡溶解约5分钟，一个充满染色槽部分。电梯机架和排出多余的蜡溶剂。 </li><li>沉浸在一个明确的仪式5分钟，第二个容器的部分。经过几次的使用，取代溶剂之间的第一和第二的容器中的第一个容器和备用。 </li><li>沉浸在100％的酒精部分，除去蜡溶剂。 </li><li>沉浸在第二个100％的酒精槽部分。 </li><li>浸入70％的酒精槽部分。 </li><li>沉浸在运行的自来水，消除酒精的所有痕迹的章节。 </li></ol> 4。缓冲液的制备<ol><li>准备磷酸盐缓冲生理盐水溶解到1公升的蒸馏水和pH值8.0 g氯化钠，氯化钾0.2克，1.15克钠2 HPO 4和0.2克的KH 2 PO 4至7.4 。 </li><li> PBS稀释到0.9倍，加入900毫升的PBS到100毫升的蒸馏水。 </li><li>准备阻止加入2毫升小鼠血清的血清200μLTRITON - X 100和500μLBSA的50毫升的0.9倍PBS。 </li><li>准备50 mM的碳酸氢盐缓冲液是由溶解在500毫升蒸馏水H 2 O 2.1克碳酸氢钠3和4 g氯化钠和滴定，pH值8.2准备。 </li></ol> 5。 Immunhistochemistry <ol><li>补充水分在0.9倍PBS浸泡的部分。 </li><li>孵育30分钟，在阻断血清的幻灯片。 </li><li>孵育Ed9（100阻断血清稀释1）在一个不透明的湿盒内室温两小时的部分。 </li><li>冲洗部分PBS中4次，每次10分钟。 </li><li>在一个不透明的湿盒内室温孵育50μL二次抗体（羊抗鼠FITC标记的抗体，阻止血清稀释500倍）在两小时的部分。 </li><li>冲洗部分缓冲液中的碳酸氢盐的3倍。 </li><li>在去离子水冲洗部分简要。 </li></ol> 6。安装<ol><li>离开幻灯片，在黑暗中干燥几分钟。 </li><li>安装使用permafluor。 </li><li>安装介质来过夜，在黑暗阴凉的环境中，干燥，优先许可。 </li></ol> 7。显微镜<ol><li>组织切片观察，荧光显微镜下。在我们的实验中，我们使用奥林巴斯BX51显微镜配备了三重（FITC; MCA得克萨斯红）过滤器。相框软件（MicroFire，Goleta，加利福尼亚州）。 </li></ol> 8。代表性的成果: 由于鱼组织的自体荧光是如此之高，这些组织会出现下脐橙的三滤，方便地提供了一个细菌的背景。 在此背景下，个体FITC染色细菌会出现明亮的绿色圆点，在200X棒状将识别（图1）。 坏的部分，可以构成假阳性（由于在冲洗阶段的问题），在这种情况下，会出现大量的细菌统一目前在幻灯片上。 <img src="/files/ftp_upload/2687/2687fig1.jpg" alt="图1"/> 图1一块肌肉的显微照片显示两个五部分。 ictaluri（箭头A和B）（200X）。 igure 2"/&gt; 图2实验的总体方案 。

<ol>
	<li>Plumb, J. A., Woo, P. T. K., Bruno, D. W. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=.">.</a> Fish Diseases and Disorders. , 479-479 (1998).</li><li>Roberts, R. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=.">.</a> Fish Pathology. , (2001).</li><li>Lawrence, M. L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=.">.</a> Louisiana State University. , (1997).</li><li>Nusbaum, K. E., Morrisson, E. E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Entry+of+35S-Labeled+Edwardsiella+ictaluri+into+Channel+Catfish.">Entry of 35S-Labeled Edwardsiella ictaluri into Channel Catfish.</a> Journal of Aquatic Animal Health. 8, 146-146 (1996).</li><li>Karsi, A., Menanteau-Ledouble, S., Lawrence, M. L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Development+of+bioluminescent+Edwardsiella+ictaluri+for+noninvasive+disease+monitoring.">Development of bioluminescent Edwardsiella ictaluri for noninvasive disease monitoring.</a> FEMS Microbiology Letters. 260, 286-286 (2006).</li><li>Miyazaki, T., Plumb, J. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Histopathology+of+Edwardsiella+ictaluri+in+channel+catfish,+Ictalurus+punctatus+(Rafinesque).">Histopathology of Edwardsiella ictaluri in channel catfish, Ictalurus punctatus (Rafinesque).</a> Journal of Fish Diseases. 8, 839-839 (1985).</li><li>Areechon, N., Plumb, J. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Pathogenesis+of+Edwardsiella+ictaluri+in+Channel+Catfish,+Ictalurus+punctatus.">Pathogenesis of Edwardsiella ictaluri in Channel Catfish, Ictalurus punctatus.</a> Journal of the World Mariculture Society. 14, 249-249 (1983).</li><li>Dumpala, P. R., Lawrence, M. L., Karsi, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Proteome+analysis+of+Edwardsiella+ictaluri.">Proteome analysis of Edwardsiella ictaluri.</a> Proteomics. 9, 1353-1353 (2009).</li><li>Thune, R. L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Signature-Tagged+Mutagenesis+of+Edwardsiella+ictaluri+Identifies+Virulence-Related+Genes,+Including+a+Salmonella+Pathogenicity+Island+2+Class+of+Type+III+Secretion+Systems.">Signature-Tagged Mutagenesis of Edwardsiella ictaluri Identifies Virulence-Related Genes, Including a Salmonella Pathogenicity Island 2 Class of Type III Secretion Systems.</a> Applied and Environmental Microbiology. 73, 7934-7934 (2007).</li><li>Karsi, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=High-throughput+bioluminescence+mutant+screening+strategy+for+identification+of+bacterial+virulence+genes.">High-throughput bioluminescence mutant screening strategy for identification of bacterial virulence genes.</a> Applied and Environmental Microbiology. 75, 2166-2166 (2009).</li><li>Hook, E. W., Mandell, G. L., Douglas, G., Bennett, J. E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=.">.</a> Principles and Practices of Infectious Diseases. , 1700-1700 (1990).</li><li>Ainsworth, J., Capley, G., Waterstreet, P., Munson, D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Use+of+monoclonal+antibodies+in+the+indirect+fluorescent+antibody+technique+(IFA)+for+the+diagnosis+of+Edwardsiella+ictaluri.">Use of monoclonal antibodies in the indirect fluorescent antibody technique (IFA) for the diagnosis of Edwardsiella ictaluri.</a> Journal of Fish Diseases. 9, 439-439 (1986).</li></ol>

该协议的细节在组织切片中的鲶鱼病原体爱德华ictaluri原位可视化技术。据我们所知，这是描述的第一个此类协议。 最关键的一步，在我们的经验，是在作为本议定书不足洗涤步骤4.4中所述的抗体冲洗，可能会导致假阳性。 这项技术的结果与解释的主要问题是组织的自体荧光。然而，人们发现，使用三滤，主要是解决问题的非特异性荧光绝大多数发生以外的绿色荧光光谱。此外，虽然这项技术是相当敏感，它可能会失败，检测细菌负荷低。

<table border="1"><tbody><tr><th>试剂名称</th><th>公司</th><th>目录编号</th></tr><tr><td>脑灌注液</td><td>碧迪</td><td> 237200 </td></tr><tr><td> MS222 </td><td>银色实验室</td><td></td></tr><tr><td>全小鼠血清</td><td> Cappel </td><td> 55989 </td></tr><tr><td>羊抗小鼠FITC </td><td>南方生物技术</td><td> 1010年至1004年</td></tr><tr><td> Permafluor </td><td>实验室的愿景</td><td> TA - 030 - FM </td></tr><tr><td>氯化钾（KCl） </td><td>西格玛</td><td> P - 4504 </td></tr><tr><td> TRITON - X </td><td> Sigma - Aldrich公司</td><td> X100 - 6X500ML </td></tr><tr><td>牛血清白蛋白</td><td>西格玛</td><td> 85040C </td></tr><tr><td>磷酸钾（KH 2 PO 4） </td><td>西格玛</td><td> P - 5379 </td></tr><tr><td>小苏打（碳酸氢钠3） </td><td>西格玛</td><td> S - 5761 </td></tr><tr><td>氯化钠（NaCal） </td><td>西格玛</td><td> S - 9625 </td></tr><tr><td>钠磷酸氢二钠（以Na 2 HPO 4） </td><td>西格玛</td><td> S - 9390 </td></tr></tbody></table>

use of fluorescent immuno-chemistry for the detection of edwardsiella ictaluri in channel catfish (i. punctatus) samples

虽然爱德华ictaluri是斑点叉尾鮰毛虫的主要病原体，并已发现了近三十年前 1,2，到目前为止，这些作者的知识，没有方法已发展到允许在原位可视化细菌在组织学切片。 虽然细菌的本地化已在以前的研究使用 3，辐射标记 4平板计数，或发光细菌体内确定，这些研究大多只进行总值器官水平，但有一个例外 6 。此限制的特别关注，是因为大肠杆菌ictaluri有一个复杂的感染周期1,7，和它具有的各种致病因素8,9。 E.复杂的相互作用与它的主机ictaluri是在许多方面类似伤寒沙门氏菌10，这是在同一分类家庭。 在这里，我们描述的一种技术，允许检测细菌用间接免疫组织化学，使用单克隆Ed9抗体安斯沃思等11。 简单地说，一个封闭血清应用于石蜡包埋组织切片，以防止非特定招标。然后，部分孵育与初级抗体:E. ictaluri特异性单克隆抗体Ed9。多余的抗体冲洗，加入FITC标记的抗体。冲洗后，部分安装带有荧光的具体安装介质。 这使得对大肠杆菌的检测ictaluri鮰组织的组织切片原位。

Watch this Scientific Journal Video about Use of Fluorescent Immuno-Chemistry for the detection of Edwardsiella ictaluri in channel catfish I. punctatus samples at JoVE.com

Use of Fluorescent Immuno-Chemistry for the detection of Edwardsiella ictaluri in channel catfish I. punctatus samples

在这里，我们描述了一个程序，允许标签<em爱德华ictaluri原位</em在斑点叉尾鮰的组织切片<em&gt;斑点叉尾毛虫</em&gt;作为一个小学，和荧光FITC标记抗体作为辅助用单克隆抗体间接免疫组化Ed9。这允许使用荧光显微镜观察细菌的检测。

使用荧光免疫化学检测爱德华ictaluri</em斑点叉尾鮰>（一毛虫）样品

While Edwardsiella ictaluri is a major pathogen of channel catfish Ictalurus punctatus and has been discovered nearly 
three decades ago 1,2, so far, to ...

use-fluorescent-immuno-chemistry-for-detection-edwardsiella-ictaluri

Nanyang Technological University

Research

JoVE Journal

Immunology and Infection

Use of Fluorescent Immuno-Chemistry for the detection of Edwardsiella ictaluri in channel catfish (I. punctatus) samples

10.4K 次观看.  Mississippi State University. 在这里，我们描述了一个程序，允许标签

视频: Use of Fluorescent Immuno-Chemistry for the detection of Edwardsiella ictaluri in channel catfish I. punctatus samples

A Time Differential Staining Technique Coupled with Full Bilateral Gill Denervation to Study Ionocytes in Fish

Branchial ionocytes (ICs) are the functional units for ionic regulation in fish. In adults, they are found on the filamental and lamellar epithelia of the gill where they transport ions such as Na+, Cl- and Ca2+ via a variety of ion channels, pumps and exchangers. The teleost gill is extrinsically innervated by the facial (VI), glossopharyngeal (IX) and vagus (X) nerves. The IX and X nerves are also the extrinsic source of branchial IC innervation. Here, two techniques used to study the innervation, proliferation and distribution of ICs are described: a time differential staining technique and a full bilateral gill denervation technique. Briefly, goldfish are exposed to a vital mitochondrion-specific dye (e.g., MitoTracker Red) which labels (red fluorescence) pre-existing ICs. Fish were either allowed to recover for 3 - 5 days or immediately underwent a full bilateral gill denervation. After 3 - 5 days of recovery, the gills are harvested and fixed for immunohistochemistry. The tissue is then stained with an &#945;-5 primary antibody (targets Na+/K+ ATPase containing cells) in conjunction with a secondary antibody that labels all (both new and pre-existing) ICs green. Using confocal imaging, it was demonstrated that pre-existing ICs appear yellow (labelled with both a viable mitochondrion-specific dye and &#945;-5) and new ICs appear green (labelled with &#945;-5 only). Both techniques used in tandem can be applied to study the innervation, proliferation and distribution of ICs on the gill filament when fish are exposed to environmental challenges.

This manuscript describes a protocol to track the re-distribution of branchial ionocytes and their innervation using a time differential staining technique coupled with full bilateral gill denervation.

一时间差别染色技术再加上双边完全失神经支配吉尔在鱼类研究Ionocytes

Branchial ionocytes (ICs) are the functional units for ionic regulation in fish. In adults, they are found on the filamental and lamellar epithelia of the ...

科研

发育生物学

Combination of Adhesive-tape-based Sampling and Fluorescence in situ Hybridization for Rapid Detection of Salmonella on Fresh Produce

This protocol describes a simple approach for adhesive-tape-based sampling of tomato and other fresh produce surfaces, followed by on-tape fluorescence in situ hybridization (FISH) for rapid culture-independent detection of Salmonella spp. Cell-charged tapes can also be placed face-down on selective agar for solid-phase enrichment prior to detection. Alternatively, low-volume liquid enrichments (liquid surface miniculture) can be performed on the surface of the tape in non-selective broth, followed by FISH and analysis via flow cytometry. To begin, sterile adhesive tape is brought into contact with fresh produce, gentle pressure is applied, and the tape is removed, physically extracting microbes present on these surfaces. Tapes are mounted sticky-side up onto glass microscope slides and the sampled cells are fixed with 10% formalin (30 min) and dehydrated using a graded ethanol series (50, 80, and 95%; 3 min each concentration). Next, cell-charged tapes are spotted with buffer containing a Salmonella-targeted DNA probe cocktail and hybridized for 15 - 30 min at 55&deg;C, followed by a brief rinse in a washing buffer to remove unbound probe. Adherent, FISH-labeled cells are then counterstained with the DNA dye 4',6-diamidino-2-phenylindole (DAPI) and results are viewed using fluorescence microscopy. For solid-phase enrichment, cell-charged tapes are placed face-down on a suitable selective agar surface and incubated to allow in situ growth of Salmonella microcolonies, followed by FISH and microscopy as described above. For liquid surface miniculture, cell-charged tapes are placed sticky side up and a silicone perfusion chamber is applied so that the tape and microscope slide form the bottom of a water-tight chamber into which a small volume (&le; 500 &mu;L) of Trypticase Soy Broth (TSB) is introduced. The inlet ports are sealed and the chambers are incubated at 35 - 37&deg;C, allowing growth-based amplification of tape-extracted microbes. Following incubation, inlet ports are unsealed, cells are detached and mixed with vigorous back and forth pipetting, harvested via centrifugation and fixed in 10% neutral buffered formalin. Finally, samples are hybridized and examined via flow cytometry to reveal the presence of Salmonella spp. As described here, our "tape-FISH" approach can provide simple and rapid sampling and detection of Salmonella on tomato surfaces. We have also used this approach for sampling other types of fresh produce, including spinach and jalape&ntilde;o peppers.

Combination of Adhesive-tape-based Sampling and Fluorescence in situ Hybridization for Rapid Detection of Salmonella on Fresh Produce

This protocol describes a simple adhesive-tape-based approach for sampling of tomato and other fresh produce surfaces, followed by rapid whole cell detection of Salmonella using fluorescence in situ hybridization (FISH).

胶粘剂基于磁带的采样和荧光组合原位杂交快速检测沙门氏菌新鲜农产品

This protocol describes a simple approach for adhesive-tape-based sampling of tomato and other fresh produce surfaces, followed by on-tape fluorescence in ...

免疫与感染

Experimental Protocol for Detecting Cyanobacteria in Liquid and Solid Samples with an Antibody Microarray Chip

Global warming and eutrophication make some aquatic ecosystems behave as true bioreactors that trigger rapid and massive cyanobacterial growth; this has relevant health and economic consequences. Many cyanobacterial strains are toxin producers, and only a few cells are necessary to induce irreparable damage to the environment. Therefore, water-body authorities and administrations require rapid and efficient early-warning systems providing reliable data to support their preventive or curative decisions. This manuscript reports an experimental protocol for the in-field detection of toxin-producing cyanobacterial strains by using an antibody microarray chip with 17 antibodies (Abs) with taxonomic resolution (CYANOCHIP). Here, a multiplex fluorescent sandwich microarray immunoassay (FSMI) for the simultaneous monitoring of 17 cyanobacterial strains frequently found blooming in freshwater ecosystems, some of them toxin producers, is described. A microarray with multiple identical replicates (up to 24) of the CYANOCHIP was printed onto a single microscope slide to simultaneously test a similar number of samples. Liquid samples can be tested either by direct incubation with the antibodies (Abs) or after cell concentration by filtration through a 1- to 3-&#956;m filter. Solid samples, such as sediments and ground rocks, are first homogenized and dispersed by a hand-held ultrasonicator in an incubation buffer. They are then filtered (5 - 20 &#956;m) to remove the coarse material, and the filtrate is incubated with Abs. Immunoreactions are revealed by a final incubation with a mixture of the 17 fluorescence-labeled Abs and are read by a portable fluorescence detector. The whole process takes around 3 h, most of it corresponding to two 1-h periods of incubation. The output is an image, where bright spots correspond to the positive detection of cyanobacterial markers.

Experimental Protocol for Detecting Cyanobacteria in Liquid and Solid Samples with an Antibody Microarray Chip

The presence of cyanobacterial toxins in fresh water reservoirs for human consumption is a major concern for water management authorities. To evaluate the risk of water contamination, this article describes an protocol for the in-field detection of cyanobacterial strains in liquid and solid samples by using an antibody microarray chip.

用于检测实验方案<em&gt;蓝藻</em&gt;与抗体基因芯片液体和固体样品

Global warming and eutrophication make some aquatic ecosystems behave as true bioreactors that trigger rapid and massive cyanobacterial growth; this has ...

环境科学

Detection of Tilapia Lake Virus Using Conventional RT-PCR and SYBR Green RT-qPCR

The aim of this method is to facilitate the fast, sensitive and specific detection of Tilapia Lake Virus (TiLV) in tilapia tissues. This protocol can be used as part of surveillance programs, biosecurity measures and in TiLV basic research laboratories. The gold standard of virus diagnostics typically involves virus isolation followed by complementary techniques such as reverse-transcription polymerase chain reaction (RT-PCR) for further verification. This can be cumbersome, time-consuming and typically requires tissue samples heavily infected with virus. The use of RT-quantitative (q)PCR in the detection of viruses is advantageous because of its quantitative nature, high sensitivity, specificity, scalability and its rapid time to result. Here, the entire method of PCR based approaches for TiLV detection is described, from tilapia organ sectioning, total ribonucleic acid (RNA) extraction using a guanidium thiocyanate-phenol-chloroform solution, RNA quantification, followed by a two-step PCR protocol entailing, complementary deoxyribonucleic acid (cDNA) synthesis and detection of TiLV by either conventional PCR or quantitative identification via qPCR using SYBR green I dye. Conventional PCR requires post-PCR steps and will simply inform about the presence of the virus. The latter approach will allow for absolute quantification of TiLV down to as little as 2 copies and thus is exceptionally useful for TiLV diagnosis in sub-clinical cases. A detailed description of the two PCR approaches, representative results from two laboratories and a thorough discussion of the critical parameters of both have been included to ensure that researchers and diagnosticians find their most suitable and applicable method of TiLV detection.

This protocol diagnoses Tilapia Lake Virus (TiLV) in tilapia tissues using RT-PCR methodologies. The entire method is described from tissue dissection to total RNA extraction, followed by cDNA synthesis and detection of TiLV by either conventional PCR or quantitative PCR using dsDNA binding a fluorescent binding dye.

常规 rt-pcr 和 sybr 绿色 rt-qpcr 检测罗非鱼

The aim of this method is to facilitate the fast, sensitive and specific detection of Tilapia Lake Virus (TiLV) in tilapia tissues. This protocol can be ...

Fluorescently Labeled Bacteria as a Tracer to Reveal Novel Pathways of Organic Carbon Flow in Aquatic Ecosystems

Elucidating trophic interactions, such as predation and its effects, is a frequent task for many researchers in ecology. The study of microbial communities has many limitations, and determining a predator, prey, and predatory rates is often difficult. Presented here is an optimized method based on the addition of fluorescently labelled prey as a tracer, which allows for reliable quantitation of the grazing rates in aquatic predatory eukaryotes and estimation of nutrient transfer to higher trophic levels.

Presented here is a protocol for a single-cell, epifluorescence microscopy-based technique to quantify grazing rates in aquatic predatory eukaryotes with high precision and taxonomic resolution.

荧光标记细菌作为追踪器揭示水生生态系统中有机碳流动的新途径

Elucidating trophic interactions, such as predation and its effects, is a frequent task for many researchers in ecology. The study of microbial ...

Characterization of a Pathogenic Escherichia coli Strain Derived from Oreochromis spp. Farms Using Whole-Genome Sequencing

Aquaculture is one of the fastest-growing food-producing sectors worldwide and tilapia (Oreochromis spp.) farming constitutes the major freshwater fish variety cultured. Because aquaculture practices are susceptible to microbial contamination derived from anthropogenic sources, extensive antibiotic usage is needed, leading to aquaculture systems becoming an important source of antibiotic-resistant and pathogenic bacteria of clinical relevance such as Escherichia coli (E. coli). Here, the antimicrobial resistance, virulence, and mobilome features of a pathogenic E. coli strain, recovered from inland farmed Oreochromis spp., were elucidated through whole-genome sequencing (WGS) and in silico analysis. Antimicrobial susceptibility testing (AST) and WGS were performed. Furthermore, phylogenetic group, serotype, multilocus sequence typing (MLST), acquired antimicrobial resistance, virulence, plasmid, and prophage content were determined using diverse available web tools. The E. coli isolate only exhibited intermediate susceptibility to ampicillin and was characterized as ONT:H21-B1-ST40 strain by WGS-based typing. Although only a single antimicrobial resistance-related gene was detected [mdf(A)], several virulence-associated genes (VAGs) from the atypical enteropathogenic E. coli (aEPEC) pathotype were identified. Additionally, the cargo of plasmid replicons from large plasmid groups and 18 prophage-associated regions were detected. In conclusion, the WGS characterization of an aEPEC isolate, recovered from a fish farm in Sinaloa, Mexico, allows insights into its pathogenic potential and the possible human health risk of consuming raw aquacultural products. It is necessary to exploit next-generation sequencing (NGS) techniques for studying environmental microorganisms and to adopt a one health framework to learn how health issues originate.

Characterization of a Pathogenic Escherichia coli Strain Derived from Oreochromis spp. Farms Using Whole-Genome Sequencing

The feasibility of whole-genome sequencing (WGS) strategies using benchtop instruments has simplified the genome interrogation of every microbe of public health relevance in a lab setting. A methodological adaptation of the workflow for bacterial WGS is described and a bioinformatics pipeline for analysis is also presented.

使用全基因组测序表征源自Oreochromis spp.农场的致病性大肠杆菌菌株

Aquaculture is one of the fastest-growing food-producing sectors worldwide and tilapia (Oreochromis spp.) farming constitutes the major freshwater fish ...

遗传学

Examination of Host Phenotypes in Gambusia affinis Following Antibiotic Treatment

The commonality of antibiotic usage in medicine means that understanding the resulting consequences to the host is vital. Antibiotics often decrease host microbiome community diversity and alter the microbial community composition. Many diseases such as antibiotic-associated enterocolitis, inflammatory bowel disease, and metabolic disorders have been linked to a disrupted microbiota. The complex interplay between host, microbiome, and antibiotics needs a tractable model for studying host-microbiome interactions. Our freshwater vertebrate fish serves as a useful model for investigating the universal aspects of mucosal microbiome structure and function as well as analyzing consequential host effects from altering the microbial community. Methods include host challenges such as infection by a known fish pathogen, exposure to fecal or soil microbes, osmotic stress, nitrate toxicity, growth analysis, and measurement of gut motility. These techniques demonstrate a flexible and useful model system for rapid determination of host phenotypes.

Examination of Host Phenotypes in Gambusia affinis Following Antibiotic Treatment

This study involves methods to reveal effects on a model fish host following alteration of the skin and gut microbiome communities&#160;composition by an antibiotic.

主机表型的考试<em&gt;食蚊鱼</em&gt;继抗生素治疗

The commonality of antibiotic usage in medicine means that understanding the resulting consequences to the host is vital. Antibiotics often decrease host ...

Using Enhanced Green Fluorescence Protein-expressing Escherichia Coli to Assess Mouse Peritoneal Macrophage Phagocytosis

This manuscript describes a simple and reproducible method to perform a phagocytosis assay. The first part of this method involves building a pET-SUMO-EGFP vector (SUMO = small ubiquitin-like modifier) and expressing enhanced green fluorescence protein (EGFP) in Escherichia coli (BL21DE). EGFP-expressing E. coli is coincubated with macrophages for 1 h at 37 &#176;C; the negative control group is incubated on ice for the same amount of time. Then, the macrophages are ready for assessment. The advantages of this technique include its simple and straightforward steps, and phagocytosis can be measured by both flow cytometer and fluorescence microscope. The EGFP-expressing E. coli are stable and display a strong fluorescence signal even after the macrophages are fixed with paraformaldehyde. This method is not only suitable for the assessment of macrophage cell lines or primary macrophages in vitro but also suitable for the evaluation of granulocyte and monocyte phagocytosis in peripheral blood mononuclear cells. The results show that the phagocytic capability of peritoneal macrophages from young (eight-week-old) mice is higher than that of macrophages from aged (16-month-old) mice. In summary, this method measures macrophage phagocytosis and is suitable for studying the innate immune system function.

Using Enhanced Green Fluorescence Protein-expressing Escherichia Coli to Assess Mouse Peritoneal Macrophage Phagocytosis

Here, we present a protocol to assess mouse peritoneal macrophage phagocytosis using enhanced green fluorescence protein-expressing Escherichia coli.

增强型绿色荧光蛋白表达大肠杆菌评价小鼠腹腔巨噬细胞吞噬细胞增多

This manuscript describes a simple and reproducible method to perform a phagocytosis assay. The first part of this method involves building a ...

Reverse Transcription Loop-Mediated Isothermal Amplification (RT-LAMP) Assay for the Specific and Rapid Detection of Tilapia Lake Virus

Tilapia lake virus disease (TiLVD), an emerging viral disease in tilapia caused by the tilapia lake virus (TiLV), is a persistent challenge in the aquaculture industry that has resulted in the mass morbidity and mortality of tilapia in many parts of the world. An effective, rapid, and accurate diagnostic assay for TiLV infection is therefore necessary to detect the initial infection and to prevent the spread of the disease in aquaculture farming. In this study, a highly sensitive and practical reverse transcription loop-mediated isothermal amplification (RT-LAMP) method is presented to detect tilapia lake virus in fish tissue. A comparison of the RT-qPCR and RT-LAMP assays of infected samples revealed positive results in 63 (100%) and 51 (80.95%) samples, respectively. Moreover, an analysis of uninfected samples showed that all 63 uninfected tissues yielded negative results for both the RT-qPCR and RT-LAMP assays. The cross-reactivity with five pathogens in tilapia was evaluated using RT-LAMP, and all the tests showed negative results. Both the liver and mucus samples obtained from infected fish showed comparable results using the RT-LAMP method, suggesting that mucus can be used in RT-LAMP as a nonlethal assay to avoid killing fish. In conclusion, the results demonstrated that the presented RT-LAMP assay provides an effective method for TiLV detection in tilapia tissue within 1 h. The method is therefore recommended as a screening tool on farms for the rapid diagnosis of TiLV.

Reverse Transcription Loop-Mediated Isothermal Amplification RT-LAMP Assay for the Specific and Rapid Detection of Tilapia Lake Virus

We present an RT-LAMP assay for the detection of TiLV in tilapia fish using simple instruments over a relatively short period of time compared to conventional RT-PCR techniques. This protocol may help control the epidemic spread of TiLVD, especially in developing countries.

反向转录循环介导的等温放大（RT-LAMP）测定，用于对罗非鱼湖病毒进行特定和快速检测

Tilapia lake virus disease (TiLVD), an emerging viral disease in tilapia caused by the tilapia lake virus (TiLV), is a persistent challenge in the ...

Development of a Colloidal Gold-based Immunochromatographic Test Strip for Detection of Cetacean Myoglobin

This protocol describes the development of a colloidal gold immunochromatographic test strip based on the sandwich format that can be used to differentiate the myoglobin (Mb) of cetaceans from that of seals and other animals. The strip provides rapid and on-the-spot screening for cetacean meat, thereby restraining its illegal trade and consumption. Two monoclonal antibodies (mAbs) with reactivity toward the Mb of cetaceans were developed. The amino acid sequences of Mb antigenic reactive regions from various animals were analyzed in order to design two synthetic peptides (a general peptide and a specific peptide) and thereafter raise the mAbs (subclass IgG1). The mAbs were selected from hybridomas screened by indirect ELISA, western blot and dot blot. CGF5H9 was specific to the Mbs of rabbits, dogs, pigs, cows, goats, and cetaceans while it showed weak to no affinity to the Mbs of chickens, tuna and seals. CSF1H13 can bind seals and cetaceans with strong affinity but showed no affinity to other animals. Cetacean samples from four families (Balaenopteridae, Delphinidae, Phocoenidae and Kogiidae) were used in this study, and the results indicated that these two mAbs have broad binding ability to Mbs from different cetaceans. These mAbs were applied on a sandwich-type colloidal gold immunochromatographic test strip. CGF5H9, which recognizes many species, was colloid gold-labeled and used as the detection antibody. CSF1H13, which was coated on the test zone, detected the presence of cetacean and seal Mbs. Muscle samples from tuna, chicken, seal, five species of terrestrial mammals and 15 species of cetaceans were tested in triplicate. All cetacean samples showed positive results and all the other samples showed negative results.

This protocol describes the development of two IgG class monoclonal antibodies (mAbs) strongly reactive to myoglobin of cetaceans. These mAbs are applied on a colloidal gold immunochromatographic test strip based on the sandwich format to differentiate the Mb of cetaceans from seal and other animals.

使用荧光免疫化学检测爱德华ictaluri（一毛虫）样品

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